Differential pH analyzers (Luzzana M., Perrella M., Rossi-Bernardi L. Anal. Biochem. 1971;43:556-563, U.S. Pat. No. 4,353,867 and EP 52718) have been extensively used to measure the concentration of chemicals in various fluids. Indeed, more than 45 papers describing the apparatus and related methods of analysis have appeared in the international literature in the last twenty years.
The essential features of a differential pH analyzer have been described in detail in the original paper and in the patents cited. The apparatus, using appropriately formulated reagents, has been used to determine a variety of substances (such as glucose, urea, creatinine, ethanol, lactate, acetate, ATP, fructose, magnesium) and the activity of several enzymes (i.e., cholinesterase, glucose-6-phosphate dehydrogenase, pyruvate kinase) in the absence or in the presence of inhibitors.
It should be noted that the key analytical equation used in differential pH measurements to calculate .DELTA.[H.sup.+ ] (i.e., the number of hydrogen ions, in moles per liter, liberated or absorbed in a chemical reaction involving the analyte to be determined) is given, under appropriate experimental conditions, by: EQU .DELTA.[H.sup.+ ]=.beta.(pH.sub.(1) -pH.sub.(0))=.beta..DELTA.pH(1)
where .beta. is the so-called "buffer value", i.e., .DELTA.[B.sup.+ ]/.DELTA.pH of the solution at any given pH and pH.sub.(0), pH.sub.(1) are the pH values of the solution before and after the chemical reaction of interest has occurred. In this context, pH is equal to -log.sub.10[H.sup.+ ].
It has been shown in the cited literature that eq. (1) can be used to determine .DELTA.[H.sup.+ ] from the known values of pH.sub.(1), pH.sub.(0) and .beta.. It should be noted that according to (1), the number of hydrogen ions produced or absorbed in a given reaction, which is directly correlated, through a known chemical reaction, to the amount of analyte present in solution, is proportional to the difference in pH and not to the absolute value of this parameter. In actual practice, the concentration of the substance of interest is obtained by the device described in U.S. Pat. No. 4,353,867 by the slightly more complicated equation: EQU [substance]=FCAL(.DELTA.pH.sub.c -.DELTA.pH.sub.b -.DELTA.pH.sub.a)=FCAL .DELTA.pH (2)
where FCAL represents a calibration factor which is a combination of .beta. and a dilution factor of the sample under analysis, .DELTA.pH.sub.c represents the total change of pH in solution after a reaction has occurred, .DELTA.pH.sub.b is the .DELTA.pH value of the solution after the addition of the reagent(s), but without the sample, and .DELTA.pH.sub.a is the .DELTA.pH value indicative of the level of noise and drift of the machine components during the observation time. Since FCAL, .DELTA.pH.sub.b, .DELTA.pH.sub.a can be determined in separate experiments and by the use of a suitable standard, eq. (2) allows the determination of the concentration of the substance of interest from the calculated value of .DELTA.pH.
It should also be stressed that to avoid in current practice measuring .beta. (the buffer value of the solution after the addition of each sample), such a buffer value has to be much higher (50-100 times higher than that of the sample) so that any change in .beta. is made practically negligible after such an addition. This, in turn, leads to the necessity of measuring the .DELTA.pH occurring in a given chemical reaction with an experimental reproducibility of 0.0001 pH units, at least twenty times greater than in the current practice of measuring pH with the already available instrumentation.
The configuration of the apparatus as described in U.S. Pat. No. 4,353,867, and therein shown in FIGS. 1-5, and particularly the simultaneous measurement of the pH difference between two glass pH electrodes respectively filled with a buffer plus sample and buffer plus sample plus a specific reagent(s) to initiate a chemical reaction (typically a small volume of an enzyme) was specially intended to increase the sensitivity and reproducibility of pH measurements In fact, by use of the previously disclosed apparatus and related methods, it has been possible:
a) to zero any unspecific pH drift which may occur (thereby obscuring the measurement of .DELTA.pH) when a biological sample (whole blood, urine, cell suspension and similar solutions or suspensions) is mixed into a buffer of different pH; PA0 b) to minimize the effect on .DELTA.pH by a change in temperature; PA0 c) to practically zero the difference in junction potential that occurs when a traditionally pH cell made by a glass pH electrode and a reference electrode are immersed respectively into a solution having a standard composition and pH and into a solution having a chemical composition and pH different from the standard. PA0 first means to transfer a suitable buffered solution, PA0 second means to transfer a sample liquid containing a substance to be determined, PA0 third means to deliver a liquid containing a reactant; PA0 a reaction chamber wherein said buffered sample liquid and said liquid containing a reactant come into contact, whereby a reaction mixture is formed, causing a pH change in solution; PA0 a first and a second microcapillary pH electrodes; PA0 means to regulate the temperature of said first and said second pH electrodes; PA0 fourth means to deliver said reaction mixture to said first pH electrode, said means comprising means to bring the temperature of said reaction mixture to the same temperature of said first pH electrode, and being at machine ground potential; PA0 fifth means to deliver said reaction mixture to said second pH electrode; PA0 electronic means for processing pH measurements made by said first and second pH electrodes and producing a visual display of the concentration of said substance to be determined. PA0 a block made of metal or another heat-conducting material in which two micro pH glass electrodes are located, said two electrodes being connected together, in parallel, by a piece of small diameter tubing of various lengths which may contain an immobilized enzyme(s) or another suitable chemical to cause the chemical reaction(s) of interest; PA0 said thermal block and the liquid flowing into the two pH electrodes being connected to a common ground, whereas the two electrode outlets are connected to a differential amplifier circuit associated with a measuring device; PA0 a heat conducting tubing, connected to electrical ground, inserted in the hydraulic circuit, between the sample and first measuring electrode so to heat a sample solution to electrode temperature; PA0 a buffer whose known composition can be varied to ensure the occurrence of a chemical reaction in conjunction with a chemical substance, for example an enzyme immobilized or confined in the tubing connecting the two pH electrodes; PA0 electronic means to convert pH electrode potentials into pH values, to program the functioning of the apparatus and to calculate, print or store, according to a known stoichiometry and related equations, the concentration of the substance(s) of interest; PA0 microperistaltic pump(s) or other suitable device(s) to move, in a series of microbore tubings and through the two measuring pH electrodes, the solution under analysis; PA0 a microdialysis tubing to sample, by diffusion through a dialysis membrane, a definite amount of the substance to be determined contained at a known concentration in an outside liquid or in an unknown concentration in a sample to be analyzed. PA0 mechanical means to change in an oscillatory mode the flow rate of the liquid in the measuring pH capillary electrodes, inside the microdialysis probe and in the tubing connecting the various parts of the apparatus so to renew the stagnant layer of liquid around the inner walls of the circuit. PA0 two microperistaltic pumps, the first pump delivering to a mixing chamber a buffer of known suitable composition, or the same buffer containing a standard concentration of the substance to be determined, or a solution containing an unknown concentration of the substance to be determined and the second pump delivering to a mixing chamber a buffer containing a chemical substance(s) such as an enzyme(s) to start the chemical reaction; PA0 a mixing chamber to effectively mix two fluids, which are continuously delivered to said mixing chamber by the two peristaltic pumps; PA0 mechanical means to oscillate the tubing coming from the mixing chamber in order to minimize the stagnant layer of liquid on the inner wall of the hydraulic circuit and of the two measuring pH electrodes; PA0 a small diameter tubing to deliver the mixed fluid into the two capillary electrodes, arranged in series and connected together through a delay tubing.
The practical use of the previously patented apparatus in a variety of analytical problems, although confirming its wide applicability, accuracy and precision, has made clear some of its limitations: the instrument has a weight of no less than 5 kg, it has to be maintained by a trained operator, it requires up to 2-3 ml of reagents for a single determination, especially to assure the complete removal from the electrodes of contamination from the previous cycle of analysis, and, finally, it requires the manual introduction of a precise volume of sample, an operation which may be critical with certain heterogeneous solutions or suspensions. As such, the analyser cannot be uses for the continuous monitoring of an industrial process or chemical parameters of biological and clinical interest in vivo (e.g., urea, creatinine, bicarbonate concentration in an artificial kidney, the determination of glucose in subcutaneous fluids during insulin or glucose perfusion etc.).